BBA - Molecular and Cell Biology of Lipids (v.1791, #4)
Editorial Board (i).
Upstream Stimulatory Factors 1 and 2 activate the human hepatic lipase promoter via E-box dependent and independent mechanisms by Diederik van Deursen; Marije van Leeuwen; Sophie Vaulont; Hans Jansen; Adrie J.M. Verhoeven (229-237).
We studied the transcriptional regulation of the HL gene by USF1 and USF2 in HepG2 cells. The transcriptional activity of the HL(− 685/+ 13) promoter construct was increased up to 25-fold by co-transfection with USF1 and USF2. Silencing of USF1 by RNA interference reduced promoter activity by 30–40%. Chromatin immunoprecipitation assays showed binding of endogenous USF1 and USF2 to the proximal HL promoter region. In gel shift assays, USF1 and USF2 bound to E-boxes at − 307/− 312 and − 510/− 516, and to the TATA-Inr region. Although the − 514C → T substitution abolished in vitro USF binding to the − 510/− 516 E-box, the increase in HL promoter activity by USF1 and USF2 was unaffected. Deletion and mutation analysis of the HL promoter region, and insertion of multiple E-box copies in front of a heterologous promoter, revealed that upregulation by USFs was mainly mediated through the − 307/− 312 E-box and the TATA-Inr region. We conclude that in HepG2 cells USF1 and USF2 regulate transcriptional activity of the HL gene through their binding to the E-box at − 307/− 312 and the TATA-Inr region.
Keywords: Hepatic lipase; LIPC; Promoter polymorphism; Transcriptional regulation; Upstream Stimulatory Factor 1; Upstream Stimulatory Factor 2;
EP3 prostanoid receptor isoforms utilize distinct mechanisms to regulate ERK 1/2 activation by Davelene D. Israel; John W. Regan (238-245).
Prostaglandin-E2 (PGE2) is a hormone derived from the metabolism of arachidonic acid whose functions include regulation of platelet aggregation, fever and smooth muscle contraction/relaxation. PGE2 mediates its physiological and pathophysiological effects through its binding to four G-protein coupled receptor subtypes, named EP1, EP2, EP3 and EP4. The EP3 prostanoid receptor is unique in that it has multiple isoforms generated by alternative mRNA splicing. These splice variants display differences in tissue expression, constitutive activity and regulation of signaling molecules. To date there are few reports identifying differential activities of EP3 receptor isoforms and their effects on gene regulation. We generated HEK cell lines expressing the human EP3-Ia, EP3-II or EP3-III isoforms. Using immunoblot analysis we found that nM concentrations of PGE2 strongly stimulated the phosphorylation of ERK 1/2 by the EP3-II and EP3-III isoforms; whereas, ERK 1/2 phosphorylation by the EP3-Ia isoform was minimal and only occurred at μM concentrations of PGE2. Furthermore, the mechanisms of the PGE2 mediated phosphorylation of ERK 1/2 by the EP3-II and EP3-III isoforms were different. Thus, PGE2 stimulation of ERK 1/2 phosphorylation by the EP3-III isoform involves activation of a Gαi/PI3K/PKC/Src and EGFR-dependent pathway; while for the EP3-II isoform it involves activation of a Gαi/Src and EGFR-dependent pathway. These differences result in unique differences in the regulation of reporter plasmid activity for the downstream effectors ELK1 and AP-1 by the EP3-II and EP3-III prostanoid receptor isoforms.
Keywords: Prostaglandin E2; E Prostanoid receptor; ERK 1/2; Protein kinase C; Phosphoinositide-3 kinase; Epidermal growth factor receptor;
Diacylglycerol kinase δ associates with receptor for activated C kinase 1, RACK1 by Shin-ichi Imai; Satoshi Yasuda; Masahiro Kai; Hideo Kanoh; Fumio Sakane (246-253).
The δ-isozyme (type II) of diacylglycerol kinase (DGK) is known to positively regulate growth factor receptor signaling. DGKδ, which is distributed to clathrin-coated vesicles, interacts with DGKδ itself, protein kinase C and AP2α. To search for additional DGKδ-interacting proteins, we screened a yeast two-hybrid cDNA library from HepG2 cells using aa 896–1097 of DGKδ as a bait. We identified aa 184–317 (WD40 repeats 5–7) of receptor for activated C kinase 1 (RACK1), which interacts with various important signaling molecules, as a novel binding partner of DGKδ. Co-immunoprecipitation analysis, using COS-7 cells co-expressing RACK1 and DGKδ, revealed that RACK1 selectively interacted with DGKδ, but not with type I DGKs, in mammalian cells. The interaction was dynamically regulated by phorbol ester. Intriguingly, DGKδ appeared to recruit RACK1 to clathrin-coated vesicles and co-localized with RACK1. These results suggest that DGKδ serves as an adaptor protein to regulate the localization of the versatile scaffold protein, RACK1.
Keywords: Diacylglycerol kinase; Receptor for activated C kinase 1; Protein kinase C; Clathrin; Diacylglycerol; Phosphatidic acid;
Fish protein hydrolysate elevates plasma bile acids and reduces visceral adipose tissue mass in rats by Bjørn Liaset; Lise Madsen; Qin Hao; Gabriel Criales; Gunnar Mellgren; Hanns-Ulrich Marschall; Philip Hallenborg; Marit Espe; Livar Frøyland; Karsten Kristiansen (254-262).
Conjugation of bile acids (BAs) to the amino acids taurine or glycine increases their solubility and promotes liver BA secretion. Supplementing diets with taurine or glycine modulates BA metabolism and enhances fecal BA excretion in rats. However, it is still unclear whether dietary proteins varying in taurine and glycine contents alter BA metabolism, and thereby modulate the recently discovered systemic effects of BAs. Here we show that rats fed a diet containing saithe fish protein hydrolysate (saithe FPH), rich in taurine and glycine, for 26 days had markedly elevated fasting plasma BA levels relative to rats fed soy protein or casein. Concomitantly, the saithe FPH fed rats had reduced liver lipids and fasting plasma TAG levels. Furthermore, visceral adipose tissue mass was reduced and expression of genes involved in fatty acid oxidation and energy expenditure was induced in perirenal/retroperitoneal adipose tissues of rats fed saithe FPH. Our results provide the first evidence that dietary protein sources with different amino acid compositions can modulate the level of plasma bile acids and our data suggest potential novel mechanisms by which dietary protein sources can affect energy metabolism.
Keywords: Bile acid; Fish protein hydrolysate; Taurine; Adipose tissue; Energy metabolism; Obesity;
Ceramide 1-phosphate inhibits serine palmitoyltransferase and blocks apoptosis in alveolar macrophages by María H. Granado; Patricia Gangoiti; Alberto Ouro; Lide Arana; Antonio Gómez-Muñoz (263-272).
We previously reported that incubation of bone-marrow derived macrophages in the absence of macrophage-colony stimulating factor (M-CSF), a cytokine that is essential for their growth and survival, resulted in stimulation of acid sphingomyelinase, accumulation of ceramides, and induction of apoptosis [A. Gomez-Munoz et al. 2004. Ceramide 1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages. J Lipid Res 45: 99–105]. Here, we show that alveolar NR8383 macrophages, which are not dependent on M-CSF for viability, undergo apoptosis when they are incubated in the absence of serum. NR8383 cells showed increased levels of ceramides under apoptotic conditions, but in contrast to bone marrow macrophage acid and neutral sphingomyelinases were only slightly activated. We found that the major mechanism for ceramide generation in NR8383 macrophages was stimulation of their synthesis de novo. This action involved activation of serine palmitoyltransferase (SPT), the key regulatory enzyme of this pathway. A relevant finding was that ceramide 1-phosphate (C1P) inhibited SPT activity and ceramide accumulation leading to inhibition of apoptosis. Furthermore, C1P enhanced the activity of antiapoptotic protein kinase B and its downstream effector nuclear factor kappa B. These observations add a new dimension to the understanding of the pro-survival actions of C1P in mammalian cells.
Keywords: Apoptosis; Ceramide 1-phosphate; Ceramide; Macrophage; Phosphatidylinositol 3-kinase; Protein kinase B; Serine palmitoyltransferase; Sphingolipid;
Suppression of adipogenesis program in cultured preadipocytes transfected stably with cyclooxygenase isoforms by Xiaoqing Chu; Li Xu; Kohji Nishimura; Mitsuo Jisaka; Tsutomu Nagaya; Fumiaki Shono; Kazushige Yokota (273-280).
Prostaglandins (PGs) are known to play a variety of roles in adipocytes and precursor cells, which have the arachidonate cyclooxygenase (COX) pathway to generate several series of PGs at different stages of life cycle of adipocytes. To gain a unique insight into the specific roles of the COX isoforms during the life cycle of adipocytes, 3T3-L1 preadipocytes were stably transfected with a mammalian expression vector harboring either cDNA coding for murine COX-1 or COX-2. The cloned stable transfectants with COX-1 or COX-2 exhibited higher expression levels of their corresponding mRNA and proteins, and greater production of PGE2 upon stimulation with free arachidonic acid or A23187 than the parent cells and the transfectants with vector only. However, either type of transfectants brought about the marked reduction in the accumulation of triacylglycerols after the standard adipogenesis program. Unexpectedly, aspirin or other COX inhibitors at different phases of life cycle of adipocytes failed to reverse the reduced storage of fats. The transfectants with COX-2 were sensitive to exogenous 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and troglitazone as peroxisome proliferator-activated receptor γ (PPARγ) agonists during the maturation phase for restoring the adipogenesis. By contrast, the transfectants with COX-1 were much less sensitive, which was reflected by much lower gene expression levels of PPARγ and the related adipocyte-specific markers. Taken together, the results suggest that the sustained overexpression of either COX-1 or COX-2 resulted in the interference of adipogenesis program through a PG-independent mechanism with a different mode of action of COX isoforms.
Keywords: Cyclooxygenase; Prostanoid; Adipocyte; 3T3-L1 cell; Stable transfection; Overexpression;
Suppression of hepatic fat accumulation by highly purified eicosapentaenoic acid prevents the progression of d-galactosamine-induced hepatitis in mice fed with a high-fat/high-sucrose diet by Satoshi Kajikawa; Tsuyoshi Harada; Akiko Kawashima; Kazunori Imada; Kiyoshi Mizuguchi (281-288).
The pathogenesis of non-alcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis. BALB/cA mice were fed with a standard diet (STD) or a high-fat and high-sucrose diet (HFHSD) for 14 days followed by intraperitoneal injection of d-galactosamine (DGalN) or vehicle. After 20–21 h, plasma and liver tissue were collected and analyzed. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in plasma were increased significantly in HFHSD-fed mice treated with DGalN compared to STD-fed mice treated with DGalN. This exacerbation by the HFHSD was also observed in the plasma soluble tumor necrosis factor receptor (sTNFR) levels, and hepatic levels of reactive oxygen species (ROS) and the fibrogenic gene expression, such as tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), connective tissue growth factor (CTGF) and osteopontin (OPN) in HFHSD-fed mice treated with DGalN. The triglyceride contents of the liver were significantly increased by the HFHSD. When eicosapentaenoic acid (EPA), a suppressor of sterol regulatory element binding protein 1 (SREBP-1), was administered to HFHSD-fed mice, the sensitivity of DGalN, as a result of plasma ALT and AST levels, was suppressed accompanied by reduced plasma sTNFR2 level and hepatic levels of triglyceride, ROS, and fibrogenic parameters, and by increased plasma adiponectin levels. These data suggest that the progression of steatotic liver injury closely depends on the accumulation of fat in the liver and is prevented by EPA through the suppression of the fatty liver change.
Keywords: Triglyceride; d-galactosamine; Alanine aminotransferase; Aspartate aminotransferase; Non-alcoholic steatohepatitis; Eicosapentaenoic acid;
Effects of docosahexaenoic acid on in vitro amyloid beta peptide 25–35 fibrillation by Michio Hashimoto; Hossain Md Shahdat; Masanori Katakura; Yoko Tanabe; Shuji Gamoh; Koji Miwa; Toshio Shimada; Osamu Shido (289-296).
Amyloid β peptide25–35 (Aβ25–35) encompasses one of the neurotoxic domains of full length Aβ1–40/42, the major proteinaceous component of amyloid deposits in Alzheimer's disease (AD). We investigated the effect of docosahexaenoic acid (DHA, 22:6, n-3), an essential brain polyunsaturated fatty acid, on the in vitro fibrillation of Aβ25–35 and found that it significantly reduced the degree of fibrillation, as shown by a decrease in the intensity of both the thioflavin T and green fluorescence in confocal microscopy. Transmission electron microscopy revealed that DHA-incubated samples were virtually devoid of structured fibrils but had an amorphous-like consistency, whereas the controls contained structured fibers of various widths and lengths. The in vitro fibrillation of Aβ25–35 appeared to be pH-dependent, with the strongest effect seen at pH 5.0. DHA inhibited fibrillation at all pHs, with the strongest effect at pH 7.4. It also significantly decreased the levels of Aβ25–35 oligomers. Nonreductive gradient gel electrophoresis revealed that the molecular size of the oligomers of Aβ25–35 was 10 kDa (equivalent to decamers of Aβ25–35) and that DHA dose-dependently reduced these decamers. These results suggest that DHA decreases the in vitro fibrillation of Aβ25–35 by inhibiting the oligomeric amyloid species and, therefore, Aβ25–35-related neurotoxicity or behavioral impairment could be restrained by DHA.
Keywords: Docosahexaenoic acid; Alzheimer's disease; Aβ25–35 fibrillation;
Serine palmitoyltransferase (SPT) deficient mice absorb less cholesterol by Zhiqiang Li; Tae-Sik Park; Yan Li; Xiaoyue Pan; Jahangir Iqbal; David Lu; Weiqing Tang; Liqing Yu; Ira J. Goldberg; M. Mahmood Hussain; Xian-Cheng Jiang (297-306).
Serine palmitoyltransferase (SPT) is the key enzyme for the biosynthesis of sphingolipids. It has been reported that oral administration of myriocin (an SPT inhibitor) decreases plasma sphingomyelin (SM) and cholesterol levels, and reduces atherosclerosis in apoE knockout (KO) mice. We studied cholesterol absorption in myriocin-treated WT or apoE KO animals and found that, after myriocin treatment, the mice absorbed significantly less cholesterol than controls, with no observable pathological changes in the small intestine. More importantly, we found that heterozygous Sptlc1 (a subunit of SPT) KO mice also absorbed significantly less cholesterol than controls. To understand the mechanism, we measured protein levels of Niemann-Pick C1-like 1 (NPC1L1), ABCG5, and ABCA1, three key factors involved in intestinal cholesterol absorption. We found that NPC1L1 and ABCA1 were decreased, whereas ABCG5 was increased in the SPT deficient small intestine. SM levels on the apical membrane were also measured and they were significantly decreased in SPT deficient mice, compared with controls. In conclusion, SPT deficiency might reduce intestinal cholesterol absorption by altering NPC1L1 and ABCG5 protein levels in the apical membranes of enterocytes through lowering apical membrane SM levels. This may be also true for ABCA1 which locates on basal membrane of enterocytes. Manipulation of SPT activity could thus provide a novel alternative treatment for dyslipidemia.
Keywords: Myriocin; SPT gene knockout; Cholesterol absorption; Dyslipidemia; Atherosclerosis;
Low concentrations of reactive γ-ketoaldehydes prime thromboxane-dependent human platelet aggregation via p38-MAPK activation by Nathalie Bernoud-Hubac; Denise Al Alam; Jennifer Lefils; Sean S. Davies; Venkataraman Amarnath; Michel Guichardant; L. Jackson Roberts; Michel Lagarde (307-313).
Oxidative stress has been strongly implicated in pathological processes. Isoketals are highly reactive γ-ketoaldehydes of the isoprostanes pathway of free radical-induced peroxidation of arachidonic acid that are analogous to cyclooxygenase-derived levuglandins. Because aldehydes, that are much less reactive than isoketals, have been shown to trigger platelet activation, we investigated the effect of one isoketal (E2-IsoK) on platelet aggregation. Isoketal potentiated aggregation and the formation of thromboxane B2 in platelets challenged with collagen at a concentration as low as 1 nM. Moreover, the potentiating effect of 1 nM isoketal on collagen-induced platelet aggregation was prevented by pyridoxamine, an effective scavenger of γ-ketoaldehydes. Furthermore, we provide evidence for the involvement of p38 mitogen-activated protein kinase in isoketal-mediated platelet priming, suggesting that isoketals may act upstream the activation of collagen-induced cytosolic phospholipase A2. Additionally, the incubation of platelets with 1 nM isoketal led to the phosphorylation of cytosolic phospholipase A2. The cytosolic phopholipase A2 inhibitors AACOCF3 and MAFP both fully prevented the increase in isoketal-mediated platelet aggregation challenged with collagen. These results indicate that isoketals could play an important role in platelet hyperfunction observed in pathological states such as atherosclerosis and thrombosis through the activation of the endogenous arachidonic acid cascade.
Keywords: Aldehyde; Stress kinase; Platelet activation; Thromboxane A2; Free radical;
Formation of molecular species of mitochondrial cardiolipin. 1. A novel transacylation mechanism to shuttle fatty acids between sn-1 and sn-2 positions of multiple phospholipid species by Ashim Malhotra; Yang Xu; Mindong Ren; Michael Schlame (314-320).
Mitochondrial cardiolipin undergoes extensive remodeling of its acyl groups to generate uniformly substituted species, such as tetralinoleoyl-cardiolipin, but the mechanism of this remodeling has not been elucidated, except for the fact that it requires tafazzin. Here we show that purified recombinant Drosophila tafazzin exchanges acyl groups between cardiolipin and phosphatidylcholine by a combination of forward and reverse transacylations. The acyl exchange is possible in the absence of phospholipase A2 because it requires only trace amounts of lysophospholipids. We show that purified tafazzin reacts with various phospholipid classes and with various acyl groups both in sn-1 and sn-2 position. Expression studies in Sf9 insect cells suggest that the effect of tafazzin on cardiolipin species is dependent on the cellular environment and not on enzymatic substrate specificity. Our data demonstrate that tafazzin catalyzes general acyl exchange between phospholipids, which raises the question whether pattern formation in cardiolipin is the result of the equilibrium distribution of acyl groups between multiple phospholipid species.
Keywords: Cardiolipin; Fatty acid; Mitochondria; Tafazzin;
Formation of molecular species of mitochondrial cardiolipin by Michael Schlame (321-325).
Formation of the unique molecular species of mitochondrial cardiolipin requires tafazzin, a transacylase that exchanges acyl groups between phospholipid molecular species without strict specificity for acyl groups, head groups, or carbon positions. However, it is not known whether phospholipid transacylations can cause the accumulation of specific fatty acids in cardiolipin. Here, a model is shown in linear algebra representation, in which acyl specificity emerges from the transacylation equilibrium of multiple molecular species, provided that different species have different free energies. The model defines the conditions and energy terms, under which transacylations may generate the characteristic composition of mitochondrial cardiolipin. It is concluded that acyl-specific cardiolipin patterns could arise from phospholipid transacylations in the tafazzin domain, even if tafazzin itself does not have substrate specificity.
Keywords: Cardiolipin; Fatty acid; Phospholipid; Tafazzin; Thermodynamics;